Spinosyns represent a novel group of macrolide compounds which have been isolated from the actinomycete Saccharopolyspora spinosa (Mertz and Yao, 1990). They are used for controlling insects (WO 97/00265, WO 94/20518, WO 93/09126, U.S. Pat. No. 5,670,364, U.S. Pat. No. 5,362,634, U.S. Pat. No. 5,227,295, U.S. Pat. No. 5,202,242). Spinosyns exhibit a strongly insecticidal but no antibacterial activity, whereby they can be distinguished from conventional macrolides such as tylosin, spiramycin and erythromycin, which have no insecticidal but antimicrobial activity.
The spinosyn structure is composed of a tetracyclic polyketide skeleton (aglycone) with a 12-membered macrolide ring and a 5,6,5-cis-anti-trans-tricycle and also a D-forosamine sugar moiety and a 2,3,4-tri-O-methyl-L-rhamnose sugar moiety (Kirst et al., 1991). More than 20 different natural spinosyn derivatives, the “A83543” complex, have hitherto been described (WO 97/00265, WO 94/20518, WO 93/09126). These derivatives differ in the substitution of one or some methyl groups on the tetracyclic skeleton, on the forosamine sugar moiety or the trimethylrhamnose sugar moiety. A 17-pseudoaglycone which lacks the forosamine sugar moiety has likewise been isolated from S. spinosa culture broths.
The main components of the A83543 complex formed by S. spinosa are the variants spinosyn A and spinosyn D which represent the essential components of the product Spinosad (cf. Pesticide Manual, British Crop Protection Council, 11th Ed., 1997, page 1272 and Dow Elanco trade magazine Down to Earth, Vol. 52, No. 1, 1997, and the references cited therein).
Based on studies on incorporating 13C-labelled acetate, propionate, butyrate or isobutyrate it was possible to show that the A83543 biosynthesis follows a polyketide biosynthesis path (Nakatsukasa et al., 1990). Polyketides are synthesized from short-chain acid building blocks such as acetate, propionate or butyrate by multifunctional enzymes, the “polyketide synthases” (PKSs). They catalyse, similar to the related fatty acid synthases (FASs), decarboxylating polycondensation steps of the building blocks activated as CoA thioesters. While FASs after each condensation step catalyse, by keto reduction, dehydration and enoyl reduction, a complete reduction of the β-oxoesters which are formed intermediately on the growing polyketide chain, PKSs can dispense with particular reduction steps. Modular type I PKSs consist of one or more large multifunctional proteins. In contrast, iterative type II PKSs are a complex composed of substantially monofunctional proteins.
The enzymatic activities of modular type I PKSs can be combined to “modules”. Here, a module carries an arrangement of three enzyme-catalytically active domains which lead to an elongation of the growing polyketide chain by a biosynthetic elongation unit. The said domains are a β-ketoacyl:acyl carrier protein synthase domain, an acyltransferase domain and a β-ketoacyl:acyl carrier protein domain. A module may also carry a ketoreductase domain, a dehydratase domain, an enoylreductase domain and a thioesterase domain. A “loading” module at the start of biosynthesis is able to carry from the said domains only an acyltransferase domain and a β-ketoacyl:acyl carrier protein domain and also an enzymatically inactive β-ketoacyl:acyl carrier protein synthase domain. A polyketide synthase domain comprises one each of the said enzymatic activities.
Owing to the potent insecticidal activity and the remarkable structure of the spinosyns, there is great interest in decoding the genetic information for their biosynthesis.